柿叶黄酮通过激活自噬与抗氧化应激协同作用改善NAFLD大鼠肝脏脂肪变性

doi:10.11958/20230145

天津医药 ›› 2023, Vol. 51 ›› Issue (11): 1211-1216.doi: 10.11958/20230145

柿叶黄酮通过激活自噬与抗氧化应激协同作用改善NAFLD大鼠肝脏脂肪变性

王莲(), 肖继椿, 田垚, 李锡晶, 王璐瑶, 王岚, 杨博涵, 张荣泉^△()

天津市医药科学研究所（邮编300020）

收稿日期:2023-02-10 修回日期:2023-05-17 出版日期:2023-11-15 发布日期:2023-11-07
通讯作者: ^△E-mail：zhangrongquan928@126.com
作者简介:王莲（1982），女，助理研究员，主要从事天然产物的提取分离与药理作用机制方面研究。E-mail：dunaine@sina.com
基金资助:
天津市卫生健康委员会天津市中医药管理局中医中西医结合科研课题(2021164);天津市卫生健康委员会天津市中医药管理局中医中西医结合科研课题(2021166)

Study of persimmon leaf flavonoids ameliorates liver steatosis by activating autophagy and antioxidant stress in NAFLD rats

WANG Lian(), XIAO Jichun, TIAN Yao, LI Xijing, WANG Luyao, WANG Lan, YANG Bohan, ZHANG Rongquan^△()

Tianjin Institute of Pharmaceutical and Medical Science, Tianjin 300020, China

Received:2023-02-10 Revised:2023-05-17 Published:2023-11-15 Online:2023-11-07
Contact: ^△E-mail：zhangrongquan928@126.com

王莲, 肖继椿, 田垚, 李锡晶, 王璐瑶, 王岚, 杨博涵, 张荣泉. 柿叶黄酮通过激活自噬与抗氧化应激协同作用改善NAFLD大鼠肝脏脂肪变性[J]. 天津医药, 2023, 51(11): 1211-1216.

WANG Lian, XIAO Jichun, TIAN Yao, LI Xijing, WANG Luyao, WANG Lan, YANG Bohan, ZHANG Rongquan. Study of persimmon leaf flavonoids ameliorates liver steatosis by activating autophagy and antioxidant stress in NAFLD rats[J]. Tianjin Medical Journal, 2023, 51(11): 1211-1216.

摘要/Abstract

摘要：

目的探讨柿叶黄酮（PLF）对非酒精性脂肪性肝病（NAFLD）大鼠的改善作用及机制。方法将雄性SD大鼠采用高脂饮食喂养12周制备NAFLD大鼠模型。将建模成功的大鼠按随机数字表法分为模型组、水林佳组（28 mg/kg）、PLF组（200 mg/kg）及PLF+自噬抑制剂组（PLF+3-MA组，200 mg/kg PLF+2 mg/kg 3-MA），另取正常SD雄性大鼠为对照组，每组6只。给药6周后，苏木素-伊红（HE）染色观察肝组织病理学改变；计算肝指数；采用全自动生化分析仪检测血清总胆固醇（TC）、三酰甘油（TG）、高密度脂蛋白胆固醇（HDL-C）、低密度脂蛋白胆固醇（LDL-C）、丙氨酸转氨酶（ALT）、天冬氨酸转氨酶（AST）；微量法测定肝脏组织中超氧化物歧化酶（SOD）和丙二醛（MDA）含量；Western blot检测肝组织核转录因子E2相关因子2（Nrf2）、自噬蛋白Sequestosome-1/Protein 62（SQSTM-1/P62）、微管相关蛋白1轻链3-Ⅱ（LC3-Ⅱ）的表达。结果对照组大鼠肝细胞形态未见明显异常，模型组肝脏细胞出现脂肪空泡和炎症反应。与对照组相比，模型组大鼠的肝指数、TC、TG、LDL-C、ALT、AST以及MDA水平均升高，P62蛋白表达增加，HDL-C和SOD水平降低，Nrf2、LC3-Ⅱ蛋白表达降低（P<0.05）。与模型组相比，水林佳组、PLF组大鼠的脂肪空泡和炎症反应明显改善，肝指数、TC、TG、LDL-C、ALT、AST和MDA水平均降低，P62蛋白表达降低，SOD水平升高，Nrf2、LC3-Ⅱ蛋白表达升高（P<0.05）。而PLF+3-MA组对大鼠血脂水平、SOD和MDA指标以及标志性蛋白的改善作用均低于PLF组。结论 PLF可通过激活自噬与抗氧化应激的协同作用对NAFLD大鼠肝脏产生保护效应。

关键词: 黄酮类, 非酒精性脂肪性肝病, 自噬, 氧化性应激, 柿叶黄酮

Abstract:

Objective To investigate the effect and mechanism of persimmon leaf flavonoids (PLF) on nonalcoholic fatty liver disease (NAFLD) in rats. Methods Male SD rats were fed the high-fat diet for 12 weeks to prepare a NAFLD rat model. The model rats were randomly divided into the model group, the shuilinjia group (28 mg/kg), the PLF group (200 mg/kg) and the PLF+autophagy inhibitor group (PLF+3-MA group, 200 mg/kg PLF+2 mg/kg 3-MA). Another normal SD male rats were selected as the control group, with 6 rats in each group. After treatment for 6 weeks, Hematoxylin-eosin (HE) staining was used to observe changes of liver morphology and pathology. The liver index was calculated. Total cholesterol (TC), triacylglycerol (TG), high density lipoprotein cholesterol (HDL-C), low density lipoprotein cholesterol (LDL-C), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) in serum were detected by automatic biochemical analyzer. The contents of superoxide dismutase (SOD) and malondialdehyde (MDA) in liver tissue were determined with microcalorimetric method. Western blot assay was performed to detect expression levels of nuclear factor erythroid 2 related factor 2 (Nrf2), autophagy protein sequencosome-1/protein 62 (SQSTM-1/P62) and microtubule associated protein 1 light chain 3-Ⅱ (LC3-Ⅱ). Results In the control group, there was no significant abnormality in the morphology of hepatocytes, while in the model group, there were fat vacuoles and inflammation in liver cells. Compared with the control group, the liver index, levels of TC, TG, LDL-C, ALT, AST and MDA were increased in the model group, and the expression level of P62 increased, while levels of HDL-C and SOD were decreased, expression levels of Nrf2 and LC3-Ⅱ decreased (P<0.05). Compared with the model group, the fat vacuoles and inflammation were significantly improved in the shuilinjia group and the PLF group. The liver index, levels of TC, TG, LDL-C, ALT, AST and MDA were decreased in the shuilinjia group and the PLF group, and the expression level of P62 decreased, while the level of SOD was increased, expression levels of Nrf2 and LC3-Ⅱ increased (P<0.05). The improvement effect on blood lipid levels, SOD and MDA indicators, as well as marker proteins were lower in the PLF+3-MA group than those of the PLF group. Conclusion PLF has protective effect on liver of NAFLD rats by activating autophagy and antioxidant stress.

Key words: flavones, non-alcoholic fatty liver disease, autophagy, oxidative stress, persimmon leaf flavonoids

中图分类号:

R575.5

图/表 8

参考文献 20

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组别	TC	TG	HDL-C	LDL-C
对照组	1.23±0.09	0.34±0.08	0.54±0.06	0.22±0.06
模型组	2.15±0.41^a	0.52±0.11^a	0.38±0.04^a	0.68±0.09^a
水林佳组	1.34±0.27^b	0.30±0.04^b	0.31±0.02^b	0.26±0.06^b
PLF组	1.04±0.21^b	0.22±0.06^b	0.31±0.03^b	0.19±0.04^bc
PLF+3-MA组	1.71±0.22^d	0.34±0.08^d	0.32±0.03	0.29±0.04^d
F	17.120^＊＊	11.963^＊＊	41.480^＊＊	67.921^＊＊

组别	TC	TG	HDL-C	LDL-C
对照组	1.23±0.09	0.34±0.08	0.54±0.06	0.22±0.06
模型组	2.15±0.41^a	0.52±0.11^a	0.38±0.04^a	0.68±0.09^a
水林佳组	1.34±0.27^b	0.30±0.04^b	0.31±0.02^b	0.26±0.06^b
PLF组	1.04±0.21^b	0.22±0.06^b	0.31±0.03^b	0.19±0.04^bc
PLF+3-MA组	1.71±0.22^d	0.34±0.08^d	0.32±0.03	0.29±0.04^d
F	17.120^＊＊	11.963^＊＊	41.480^＊＊	67.921^＊＊

组别	ALT/（U/L）	AST/（U/L）	肝指数/%
对照组	54.48±3.50	119.78±10.46	2.64±0.31
模型组	74.75±5.09^a	176.87±12.94^a	4.48±0.83^a
水林佳组	54.58±5.00^b	131.42±10.60^b	2.72±0.29^b
PLF组	48.92±3.57^bc	102.17±10.60^bc	2.69±0.36^b
PLF+3-MA组	51.72±3.45	140.45±10.20^d	2.73±0.19
F	35.934^＊＊	38.405^＊＊	18.487^＊＊

组别	ALT/（U/L）	AST/（U/L）	肝指数/%
对照组	54.48±3.50	119.78±10.46	2.64±0.31
模型组	74.75±5.09^a	176.87±12.94^a	4.48±0.83^a
水林佳组	54.58±5.00^b	131.42±10.60^b	2.72±0.29^b
PLF组	48.92±3.57^bc	102.17±10.60^bc	2.69±0.36^b
PLF+3-MA组	51.72±3.45	140.45±10.20^d	2.73±0.19
F	35.934^＊＊	38.405^＊＊	18.487^＊＊

组别	SOD/（U/g）	MDA/（nmol/g）
对照组	106.91±9.50	23.99±2.99
模型组	39.13±7.07^a	98.33±9.76^a
水林佳组	75.85±8.62^b	36.34±5.76^b
PLF组	101.17±10.22^bc	31.68±5.45^b
PLF+3-MA组	53.33±7.81^d	44.10±6.43^d
F	68.246^＊＊	126.744^＊＊

柿叶黄酮通过激活自噬与抗氧化应激协同作用改善NAFLD大鼠肝脏脂肪变性

Study of persimmon leaf flavonoids ameliorates liver steatosis by activating autophagy and antioxidant stress in NAFLD rats

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组别	Nrf2	P62	LC3-Ⅱ
对照组	2.17±0.13	0.87±0.06	1.95±0.15
模型组	0.76±0.02^a	2.85±0.18^a	1.03±0.09^a
水林佳组	1.40±0.07^b	1.24±0.08^b	1.65±0.06^b
PLF组	3.89±0.29^bc	1.03±0.08^bc	1.66±0.08^b
PLF+3-MA组	1.47±0.05^d	1.55±0.07^d	1.23±0.12^d
F	407.064^＊＊	337.128^＊＊	77.051^＊＊

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